1. Field of the Invention
This invention relates to a reagent for selectively precipitating low density lipoproteins, very low density lipoproteins and other non-high density lipoproteins in serum.
2. Description of the Prior Art
Considerable attention has been given to the determination of cholesterol in the high-density lipoprotein (HDL) fraction of human serum. In 1951, Barr et al.sup.1 published the results of a study indicating a definite relationship between levels of HDL and the occurrence of coronary heart disease. Since then, further research.sup.2,3,4 has provided strong evidence of a negative correlation between HDL-cholesterol levels and the risk of coronary heart disease in both men and women over the age of 50. In particular, the well-documented epidemiologic data from the National Institute of Health (NIH)-sponsored studies,.sup.5,6,7 have established an inverse correlation between decreased levels of HDL-cholesterol and premature heart disease. Studies done in other countries have supported these conclusions..sup.8,9
Determination of HDL-cholesterol has, until recently, been a difficult assay to perform. For many years, ultracentrifugation was the only acceptable means of separating the various lipoprotein fractions of human serum..sup.10 The high cost of the equipment and the time required for this type of analysis have severely restricted the application of this methodology in the clinical laboratory. Electrophoresis on various support media.sup.11 offered a simpler approach for the separation of lipoprotein fractions but did not permit ready quantitation of the lipids in the separated fractions. More recently a number of simpler methods have been developed for the isolation of plasma HDL by selective precipitation of the other lipoprotein fractions.sup.12,13,14,15. The Lipid Research Clinic (LRC) of the NIH has evaluated these procedures and proposed a standardized method in which cholesterol is measured colorimetrically (modified Lieberman-Burchard technique) in the HDL fraction obtained after precipitation of low-density lipoproteins (LDL) and very low density lipoproteins (VLDL) from whole serum with heparin/manganese..sup.16
Although extensively used today, this method represents serious problems in terms of its routine use in the clinical laboratory. Heparin preparations vary considerably from one manufacturer to another and even from lot to lot. Thus the laboratory using this procedure must verify the completeness of LDL and VLDL precipitations each time a different lot of heparin is used. The Lieberman-Burchard procedure has major drawbacks in the area of specificity and accuracy and requires the use of corrosive and noxious chemicals. To improve the specificity and avoid the use of dangerous chemicals, the heparin/manganese precipitation proposed by LRC has been coupled with the enzymatic determination of cholesterol in the HDL supernatant thus obtained. This combination has presented special problems of its own, such as formation of a visible precipitate with the enzymatic reagent and falsely increased cholesterol values..sup.17
A method employing dextran sulfate and magnesium to precipitate LDL, VLDL, and other non-HDL from whole serum or plasma has been shown to precipitate quantitatively all the VLDL and LDL in human serum and to yield accurate and reproducible values for HDL cholesterol in combination with the enzymatic cholesterol procedure..sup.18 These precipitated LDL, VLDL, and other non-HDL fractions are removed by centrifugation. The cholesterol in the HDL remaining in the supernatant is quantitated by means of the following enzyme reactions. EQU Cholesterol Esters .sup.CE* Cholesterol+Fatty Acids EQU Cholesterol+O.sub.2 .sup.CO** Cholesten-3-one+H.sub.2 O.sub.2 EQU 2H.sub.2 O.sub.2 +4 Aminoantipyrine+Phenol .sup.HPO*** Quinoneimine+2H.sub.2 O FNT *CE=cholesterol esterase FNT **Cholesterol oxidase FNT ***HPO=Peroxidase
The quinoneimine produced has an absorbance maximum at 500 nm. The intensity of the color produced is directly proportional to the concentration of cholesterol in the HDL fraction.
With very lipemic serum it is very difficult to perform the above mentioned centrifugation step required to remove the precipitated LDL, VLDL, and other non-HDL fractions from the supernatant. Therefore, in the case of very lipemic serum, it is necessary to either dilute the serum prior to the precipitation step or filter the supernatant subsequent to the centrifugation step to avoid falsely elevated HDL cholesterol values.
It would be very advantageous if one could avoid having to perform these additional steps required to circumvent the problem posed when very lipemic serum is assayed for HDL cholesterol.
It is an object of the instant invention to provide a non-HDL precipitant which can be employed without the necessity of having to perform either of the two auxiliary steps previously found necessary in order to obtain accurate HDL cholesterol values when assaying very lipemic serum.
The object of the instant invention is accomplished by adding a suitable inert, insoluble, adsorbtive composition to a reagent comprising a precipitator capable of precipitating LDL, VLDL, and other non-HDL fractions.